The ubiquitous polyomavirus JC virus may be reactivated during prolonged immune suppression from a latent infection in kidney or other organs to a productive infection of oligodendrocytes known as PML. Events leading to brain pathology are not known but may include or result in alteration of the regulatory region. The kidneys of six PML patients were examined by PCR amplification with three primer pairs for the presence of JC virus and three were positive. The use of a PCR-based typing assay on all tissue samples, and cloned sequences from the viral coding region from each positive kidney, showed that the same viral genome was present in the kidney as in the brain of the patient. Regulatory region clones all had the archetypal promoter~enhancer structure. However, when PCR fragments from the regulatory region were digested with a restriction enzyme which cuts in region D, the region most often deleted in PML-type promoters, a low level of undigested DNA remained. This DNA refractory to digestion had a rearranged sequence identical to that of the uniquely rearranged promoter in the brain of each patient. The archetype promoter/enhancer and three pro/enh obtained from PML brains representing increasing degrees of rearrangement of the archetypal pro/enh sequence were placed in front of the CAT reporter gene to assess transcriptional activity. In glial cells, the archetypal pro/enh is active in both early and late directions, and is comparable in strength to PML-derived regulatory regions. The moderately rearranged promoter, as well as the laboratory strain Mad-1 which is of the same rearrangement pattern, were the most efficient and showed the most stimulation of the late pro/enh by T-antigen. The minimally and highly rearranged regulatory regions were less efficient than the archetype. Differences in relative early to late promoter strength may be significant in JCV's ability to propagate in different cell types.